Project Summary Rotator cuff tendon injury is a very most common musculoskeletal injury, with over 3 million injuries being treated in the United States and European Union each year. These injuries have limited healing and repair potential due to poor vascularization, preventing adequate delivery of nutrients and tendon cells to produce new tissue. Recently, many tissue engineering approaches have been introduced to repair rotator cuff injuries. However, not many products have succeeded due to the lack of the currently available biomaterials. To further development in this area and overcome limitations with current devices, we propose a biomimetic matrix based-multiphasic scaffold designed to be used in conjunction with existing suture anchors to enhance repair and regeneration of the rotator cuff enthesis. In this proposal, further, we will perform in vitro evaluations of cytocompatibility of the scaffolds, and evaluate the in vivo performance of the scaffolds using a small animal rotator cuff repair model. Aim 1 will determine optimal multiphasic scaffold parameters to promote zone- specific enthesis tissue formation in rotator cuff. Aim 2 will evaluate the biological performances of zone- specific multiphasic scaffolds using a co-culture model and an animal rotator cuff repair model. This data will provide valuable references for our future design of a large animal model for translational research and commercialization. Finally, this study will advance a novel multiphasic biomaterial scaffold towards clinical implementation for treatment of rotator cuff tears with the potential for rapid translation to clinical trials and practice.